Aakash Parikh

Lab report 2 Mr. Veres 11/23/2011 Factors influencing Enzyme activity Objective: To see the changes in enzymatic activity through a series of test and condition , under which cellular respiration would occur most. Seeing how different stimuli can affect the outcome of how an enzyme works to catalyze a reaction; whether it will speed the reaction up immensely or slightly. Materials: Water tub 100mL graduate cylinder Ring stand Thermometer Reaction chamber Yeast ( Catalase) 10mL graduated cylinder Pipettes 3% hydrogen peroxide Hot plate Ice pH buffer 4,7,10 balance Distilled Water NaCl (Salt)

Introduction: Enzymes are a key part to biological functions in organism. Every organism performs a series of various chemical reactions every day. These reactions helps the human body function. Normally, these reactions would process very slow and it would years for the body to break down glucose because there are catalysts that help slow down the reaction. Biological catalysts are called enzymes. In this lab we discuss how these different stimuli actually affected the way the yeast solution respired. Hypothesis:

Part A) If 10 mL of hydrogen peroxide (H2O2) is combined in a reaction chamber with 1.0 mL of stock catalase solution (yeast) and the reaction rate is measured via the production of oxygen gas over the course of five minutes, then a control group will be established by which environmental factors can be analyzed for their influence on enzymatic activity.

Part B) If 10 mL of hydrogen peroxide (H2O2) is combined in a reaction chamber with varying concentrations of stock catalase solution (yeast) separately and the reaction rate is measured via the production of oxygen gas over the course of five minutes, then the reaction rate will increase as enzyme concentration increases because there are more active sites for which the substrate molecules can bind. Ultimately, the reaction rate will level off because the substrate is limiting.

Part C) If 10 mL of hydrogen peroxide (H2O2) is combined in a reaction chamber with 1.0 mL of stock catalase solution (yeast) at varying temperatures and the reaction rate is measured via the production of oxygen gas over the course of five minutes, then the reaction rate will increase as the enzyme approaches its optimal temperature and decrease as the enzyme moves away from its optimal temperature.

Part D) If 10 mL of hydrogen peroxide (H2O2) at three pH values is separately combined in a reaction chamber with 1.0 mL of stock catalase solution (yeast) and the reaction rate

is measured via the production of oxygen gas over the course of five minutes, then the reaction rate will increase as the enzyme approaches its optimal pH and decrease as the enzyme deviates from its optimal pH.

Part E) If 10 mL of hydrogen peroxide (H2O2) at varying concentrations is separately combined in a reaction chamber with 1.0 mL of stock catalase solution (yeast) and the reaction rate is measured via the production of oxygen gas over the course of five minutes, then the reaction rate will increase as substrate concentration increases because there will be more opportunities for the substrate molecules to bind to the active sites. Ultimately, the reaction rate will level off because all of the active sites will be filled.

Part F) If 5 mL of hydrogen peroxide (H2O2) is mixed with 5 mL of varying concentrations of NaCl independently, combined in a reaction chamber with 1.0 mL of stock catalase solution (yeast), and measured for the reaction rate via the production of oxygen gas over the course of five minutes, then the reaction rate will increase as the enzyme approaches its optimal salinity and decrease as the enzyme diverges from its optimal salinity. II. Procedure: Part A) The Time Course of Enzyme Activity 1. Fill a plastic rectangular container full of tap water. Submerge the graduated cylinder to fill most of it with water. Turn the graduated cylinder upside down to keep the water in. Suspend it in the clamp on the ring stand. Adjust the height of the clamp on the ring stand so the open end of the graduated cylinder is about 3 cm above the bottom of the container. Record the temperature of the water. Refer to the labeled diagram to the right for concerns or questions, but do not submerge the reaction chamber yet. 2. Obtain a reaction chamber, 10 mL of 3% hydrogen peroxide (H2O2) solution, and some stock catalase solution (yeast). 3. Pour the 10 mL of hydrogen peroxide (H2O2) into the reaction chamber. Pipette in 1.0 mL of stock catalase solution (yeast). Stopper the reaction chamber tightly, submerge it in the water bath, and place the plastic tubing into the bottom of the graduated

cylinder so that the bubbles formed in the reaction chamber are captured by the inverted graduated cylinder. 4. Measure the gas level in the graduated cylinder initially and at 30-second intervals for five minutes. The data should be plotted on a graph. Part B) The Effect of Enzyme Concentration on Enzyme Activity 1. Repeat Part A, but this time use three different levels of enzyme concentration as follows: - 75% concentration: Pipette in 0.75 mL of catalase solution in the reaction chamber instead of 1.0 mL. - 50% concentration: Pipette in 0.50 mL of catalase solution in the reaction chamber instead of 1.0 mL. - 25% concentration: Pipette in 0.25 mL of catalase solution in the reaction chamber instead of 1.0 mL. 2. Data recorded from the varying enzyme concentrations should be plotted on a graph. Part C) The Effect of Temperature on Enzyme Activity 1. Repeat Part A, but this time use three different temperatures as follows: - 5C: Set up your water bath and add ice so that it is chilled to 5C for five minutes before running the experiment. Keep adding ice to maintain the cool temperature. - 37C: Set up your water bath with heated water so that it is warmed to 37C for five minutes before running the experiment. Keep adding hot water to maintain the warm temperature. - 100C: Rather than using a water bath of boiling water, boil the catalase solution for five minutes. Let the catalase cool and then run the experiment in room temperature water. 2. Data recorded from the varying temperatures should be plotted on a graph. Part D) The Effect of pH on Enzyme Activity 1. Repeat Part A, but this time use 1.5% hydrogen peroxide solutions at three different pH values as follows: - pH 4: Add 5 mL of H2O2 to 5 mL of pH 4 buffer. - pH 7: Add 5 mL of H2O2 to 5 mL of pH 7 buffer. - pH 10: Add 5 mL of H2O2 to 5 mL of pH 10 buffer.

2. Data recorded from the solutions at varying pH values should be plotted on a graph. Part E) The Effect of Substrate Concentration on Enzyme Activity 1. Repeat Part A, but this time use four different substrate concentrations as follows: - 0%: Use 10 mL of distilled water only. - 0.3%: Prepare this by adding 3 mL of H2O2 to 7 mL of distilled water. - 1.5%: Prepare this by adding 5 mL of H2O2 to 5 mL of distilled water. - 3.0%: Use the data from Part A. 2. Data recorded from the varying substrate concentrations should be plotted on a graph. Part F) The Effect of Ionic Concentration on Enzyme Activity 1. Repeat Part A, but this time mix 5 mL of hydrogen peroxide solution with three different ionic concentrations as follows: - 10% NaCl: Dissolve 5 g of NaCl in 50 mL of water and add this solution to 5 mL of H2O2. - 2% NaCl: Dissolve 1 g of NaCl in 50 mL of water and add this solution to 5 mL of H2O2. - 0% NaCl: Add 5 mL of distilled water to 5 mL of H2O2.

Data

Part A and B
1 mL of yeast: 30 sec: 4mL 1 min: 6mL 1.30: 9mL 2 min: 12mL 2.30: 15.5 mL 3 min: 18 mL 3.30: 21 mL 4 min: 22 mL 4.30: 24.5 mL 5 min: 26 mL .75 ml of yeast: 30 sec: 1mL 1 min: 4mL 1.30: 5.5mL 2 min: 7mL 2.30: 11 mL 3 min: 16 mL 3.30: 20 mL 4 min: 22 mL 4.30: 23.5 mL 5 min: 24.5 mL 5 ml of yeast: 30 sec: 1mL 1 min: 3mL 1.30: 4mL 2 min: 4.5 mL 2.30: 4.7 mL 3 min: 5 mL 3.30: 5.5 mL 4 min: 5.7 mL 4.30: 6 mL 5 min: 6.3 mL .25 mL of yeast 30 sec: .7 mL 1 min: 1 mL 1.30: 1.3 mL 2 min: 1.5 mL 2.30: 1.8 mL 3 min: 2 mL 3.30: 2.2 mL 4 min: 2.4 mL 4.30: 2.6 mL 5 min: 2.7 mL

Part C
5* C 30 sec: .1mL 1 min: .1 mL 1.30: .2 mL 2 min: .2 mL 2.30: .2 mL 3 min: .3 mL 3.30: .3 mL 4 min: .4 mL 4.30: .4 mL 5 min: .5 mL

37*C 30 sec: 2 mL 1 min: 5 mL 1.30: 7.3 mL 2 min: 8.4 mL 2.30: 13 mL 3 min: 15 mL 3.30: 17 mL 4 min: 19.4 mL 4.30: 20.2 mL 5 min: 22.4 mL

100*C 30 sec: 1 mL 1 min: 1.5 mL 1.30: 1.8 mL 2 min: 1.9 mL 2.30: 2.0 mL 3 min: 2.1 mL 3.30: 2.2 mL 4 min: 2.3 mL 4.30: 2.4 mL 5 min: 2.5 mL

Part D
PH 4 30 sec: 2 mL 1 min: 3.5 mL 1.30: 4.1 mL 2 min: 4.7 mL 2.30: 5.1 mL 3 min: 5.3 mL 3.30: 5.6 mL 4 min: 5.8 mL 4.30: 6 mL 5 min: 6.3 mL

PH 7 30 sec: 2 mL 1 min: 3.9 mL 1.30: 5 mL 2 min: 5.9 mL 2.30: 6.1 mL 3 min: 6.8 mL 3.30: 7.3 mL 4 min: 7.9 mL 4.30: 8.4 mL 5 min: 9 mL

PH 10 30 sec: 1 mL 1 min: 1.5 mL 1.30: 1.9 mL 2 min: 2.1 mL 2.30: 2.3 mL 3 min: 2.7 mL 3.30: 2.9 mL 4 min: 3 mL 4.30: 3.2 mL 5 min: 3.3 mL

Part E 0% substrate
30 sec: 2 mL 1 min: 3 mL 1.30: 3.7 mL 2 min: 4 mL 2.30: 4.2 mL 3 min: 4.3 mL 3.30: 4.3 mL 4 min: 4.4 mL 4.30: 4.5 mL 5 min: 4.5 mL

.3% substrate
30 sec: 2.3 mL 1 min: 4.7 mL 1.30: 7 mL 2 min: 9 mL 2.30: 11 mL 3 min: 13 mL 3.30: 15 mL 4 min: 15.7 mL 4.30: 17 mL 5 min: 19 mL

1.5% substrate
30 sec: 2.5 mL 1 min: 6 mL 1.30: 9 mL 2 min: 12 mL 2.30: 14.3 mL 3 min: 6.2 mL 3.30: 18.1 mL 4 min: 19.3 mL 4.30: 21 mL 5 min: 23.4 mL

3% substrate
30 sec: 4mL 1 min: 6mL 1.30: 9mL 2 min: 12mL 2.30: 15.5 mL 3 min: 18 mL 3.30: 21 mL 4 min: 22 mL 4.30: 24.5 mL 5 min: 26 mL

Part F 10% NaCl

2% NaCl
30 sec: 5mL 1 min: 8.5mL 1.30: 13.5mL 2 min: 17mL 2.30: 20 mL 3 min: 23 mL 3.30: 26 mL 4 min: 29 mL 4.30: 31 mL 5 min: 35 mL

0% NaCl
30 sec: 2.5 mL 1 min: 6 mL 1.30: 9 mL 2 min: 12 mL 2.30: 14.3 mL 3 min: 6.2 mL 3.30: 18.1 mL 4 min: 19.3 mL 4.30: 21 mL 5 min: 23.4 mL

Conclusion: Part A) The increase in the gas level or the amount of O2 evolved was 8 mL over the course of five minutes. The reaction rate, which is the change in gas level divided by the change in time for this part of the laboratory and all future parts, is the control group for enzyme concentration (100%), temperature (24C), and substrate concentration (3.0%). This part of the laboratory allows us to introduce environmental conditions, or independent variables, in future parts and conclude how they affect the rate of enzyme activity. Part B) For 100% enzyme concentration, the increase in the gas level or the amount of O2 evolved was 8 mL over the course of five minutes. For 75% enzyme concentration, the increase in the gas level or the amount of O2 evolved was 6 mL over the course of five minutes. For 50% enzyme concentration, the increase in the gas level or the amount of O2 evolved was 5 mL over the course of five minutes. For 25% enzyme concentration, the increase in the gas level or the amount of O2 evolved was 2 mL over the course of five minutes. The enzyme concentration is directly proportional to the rate of enzyme activity. In laymans terms, as the enzyme concentration increases, the rate of the enzyme activity does as well. Part C) For the reaction at 24C, the increase in the gas level or the amount of O2 evolved was 8 mL over the course of five minutes. For the reaction at 5C, the increase in the gas level or the amount of O2 evolved was 2 mL over the course of five minutes. For the reaction at 37C, the increase in the gas level or the amount of O2 evolved was 20 mL over the course of five minutes. For the reaction at 100C, the increase in the gas level or the amount of O2 evolved was 2 mL over the course of five minutes.

 There is no direct correlation between temperature and the rate of enzyme activity. Ergo, the stock catalase solution (yeast) has an optimal temperature at which it operates best. Based on the data, the optimal temperature must be somewhere in the range of 37C. Part D) For the reaction at the pH of 4, the increase in the gas level or the amount of O2 evolved was 10 mL over the course of five minutes. For the reaction at the pH of 7, the increase in the gas level or the amount of O2 evolved was 8 mL over the course of five minutes. For the reaction at the pH of 10, the increase in the gas level or the amount of O2 evolved was 5 mL over the course of five minutes. The pH value is inversely proportional to the rate of enzyme activity. In laymans terms, as the pH value increases (more basic), the rate of enzyme activity decreases. Keep in mind, however, that the full range of pH values was not tested. Therefore, there may be no correlation between pH value and the rate of enzyme activity. Instead, there could be an optimal pH value somewhere in the range of pH 4. Part E) For 3.0% substrate concentration, the increase in the gas level or the amount of O2 evolved was 8 mL over the course of five minutes. For 0% substrate concentration, the increase in the gas level or the amount of O2 evolved was 0 mL over the course of five minutes. This was because there was no substrate in the form of hydrogen peroxide to decompose into oxygen gas. For 0.3% substrate concentration, the increase in the gas level or the amount of O2 evolved was 7 mL over the course of five minutes. For 1.5% substrate concentration, the increase in the gas level or the amount of O2 evolved was 8 mL over the course of five minutes. The substrate concentration is directly proportional to the rate of enzyme activity. In laymans terms, as the substrate concentration increases, the rate of enzyme activity does as well.

 However, the reaction rate for 1.5% substrate concentration and 3.0% substrate concentration are equal because the total number of enzymes and active sites becomes a limiting factor. Part F) For 10% NaCl concentration, the increase in the gas level or the amount of O2 evolved was 3 mL over the course of five minutes. For 2% NaCl concentration, the increase in the gas level or the amount of O2 evolved was 9 mL over the course of five minutes. For 0% NaCl concentration, the increase in the gas level or the amount of O2 evolved was 10 mL over the course of five minutes. The ionic concentration, or salinity, is inversely proportional to the rate of enzyme activity. In laymans terms, as the salinity increases, the rate of the enzyme activity decreases. Keep in mind, however, that the full range of salinities was not tested. Therefore, there may be no correlation between salinity and the rate of enzyme activity. Moreover, the reaction rate of 2% and 0% are very close to one another. There could be an optimal salinity somewhere in the range of 0-2%.